In this study we examined the lymph nodes of Stage II colorectal cancer patients to identify CD4+, CD8+ and Foxp3+ cell populations and correlated these with patient outcome, alone, and in combination with other clinico-pathological variables. Methods Patients Patients with UICC stage II colon cancer were included in this study. Stage II patients were chosen because they have no tumour metastases in lymph nodes. The number of lymph nodes retrieved from patients for staging is indicated in

Table 1. Approximately 50% of the lymph nodes obtained from each patient were randomly selected for immunohistochemical analysis. Table 1 Clinical characteristics of patients     CRC – recurrent CRC – non recurrent IBD controls Number patients   13 18 9 Age (years, mean (SD))   70.84 (8.922) 72.24 (11.032)   Gender %            M   39 28      F   61 72   Differentiation Poor 1 3     Moderate CHIR-99021 order 11 14     Well 1 1   Tumour Site Right 8 13     Left 5 2     Rectum 0 1   Number lymph nodes used for staging (mean (SD))   20 (12) 19 (8)   Number lymph nodes analysed (mean (SD))   10 (6) 11 (8) 5 (3) All patients underwent elective surgery for colon cancer at Dunedin Hospital, New Zealand. Pathological staging was verified by the study pathologist (HSY). In addition to colon cancer, patients

with inflammatory bowel disease were used as controls. The study was approved by buy OSI-027 the Lower South Regional

Ethics Committee and patients gave signed informed consent to participate. All patients were prospectively followed up for a minimum of five years from the date of surgery. Immunohistochemical Analysis Formalin fixed paraffin embedded (FFPE) lymph nodes recovered at surgery were used for immunostaining. 4 um serial sections were stained for T cell markers using two methods. Tonsil tissues were used as positive and negative controls. CD4 and CD8 Sections were dried for 30 min after cutting, then dewaxed on the Bond™ (Leica Microsystems, Germany) after manual drying. Heat induced epitope retrieval Celastrol was performed using ER2 (Bond™) at pH 9.0 for 20 min at 100°C. After blocking with 3% peroxide block for 5 min, the sections were incubated with the specific antibody (anti-human CD4 (NCL-L-CD4-368; Novocastra, Leico Microsystems; 1:40 dilution) or anti-human CD8 (NCL-CD8-4B11; Novocastra, Leico Microsystems; 1:100 dilution)) for 20 min at RT. Unbound antibody was removed by 3 washes in Bond™ Wash Solution before adding polymer for 10 min at RT. After washing unbound labeled polymer in Bond™ Wash Solution 3 times, peroxidase staining in tissue sections was revealed by DAB solution (Bond™). After stopping the reaction in running water, sections were counter-stained with a rinse in hematoxylin solution. After dehydration, the sections were mounted with DPX.